A microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease amplification for selective and sensitive detection of miRNAs in cancer cells

MicroRNAs (miRNAs) are considered as the potential biomarkers for many cancers. To determine miRNAs in cancer cells is significant for realizing these diseases. In this work, a microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease (DSN) amplification was developed and applied to selectively and sensitively determine miRNAs in cancer cells. An interface for laser-induced fluorescence detection was firstly applied to perform the sample detection on the paper-based chip. Under the optimal conditions, DSN (3 mu L 0.10 U) and Taqman probes (2 mu L 2.5 x 10(-7) M) were preserved on the circles (Diameter 4 mm) of the folded paper chip. When miRNA solution was added, the mixed solution could trigger fluorescence signal amplification by cyclically digesting hybrids of miRNAs and Taqman probes by DSN. The whole determination, including sample heating process, could be accomplished within 40 min. The detection limits for miRNA-21 and miRNA-31 were 0.20 and 0.50 fM respectively, corresponding to only 1.0 and 1.5 zmol consumption of miRNAs. The testing of mismatched miRNAs showed that the method had good specificity. Finally, the method was applied to determine miRNA-21 and miRNA-31 in lysates of cancer cells of A549 and HeLa, and hepatocyte LO2. MiRNA-21 and miRNA-31 could be successfully found from the two cancer cells. The concentrations for miRNA-21 and miRNA-31 were 1.74 x 10(-13) M and 6.29 x 10(-14) M in HeLa cell lysate (3.75 x 10(4) cells/mL), 3.07 x 10(-15) M and 3.28 x 10(-15) M in A549 cell lysate (8.33 x 10(6) cells/mL) respectively. The recoveries ranged from 87.30% to 111.83%, indicating the results were reliable. The developed method was effective, selective and sensitive in the determination of miRNAs in cancer cells.